The long-term goal of this laboratory is to understand how Japanese encephalitis virus (JEV) gains access to the interior of human cells, the first step in an infection process that involves a cascade of multiple, highly coordinated interactions between the virus and its target cells. This aspect of virus research is of particular relevance because virus entry is a common feature that is essential to the initiation, dissemination, and maintenance of productive infection by all human and animal viruses. While the virus-derived factors involved in JEV entry are well known (particularly viral glycoprotein E, which attaches the virions to the cell surface, delivers them to endosomes, and catalyzes the viral-endosome membrane fusion), the host factors that participate in this multistep process remain elusive. In particular, identification of the cell-surface receptor(s) for JEV entry has been a major challenge in JEV biology, primarily due to a lack of availability of non-susceptible cells, which are require for receptor screening. We have now identified a non-susceptible non-human cell line that has a block in JEV entry but fully supports the subsequent post-entry steps; this cell line remains highly susceptible to infection by two other enveloped RNA viruses which, like JEV, enter the cells through clathrin-dependent endocytic pathway. This cell line offers us a unique opportunity to identify and validate the host factors involved in JEV entry. JEV is a mosquito-borne flavivirus that is closely related to West Nile virus, St. Louis encephalitis virus, and Murray Valley encephalitis virus, all of which cause fatal neurological disease in humans. The work proposed here involves the use of two complementary, technologically advanced genome- scale genetic screens for gain- and loss-of-function of JEV entry (Aims 1 and 2, respectively) to identify the cellular factors and mechanisms in the human host that are critical for viral entry and to dissect the discrete entry steps that are regulated by specific host factors. Two independent but complementary aims will be carried out: In Aim 1, an iterative cDNA library screening approach will be used to identify one or more cell type-specific cellular genes in highly JEV-susceptible non-neuronal and neuronal human cells that confer susceptibility to JEV infection on a newly developed, JEV-non-susceptible non-human cell line. In Aim 2, a multiplexed RNAi screening approach will be employed to identify one or more cell type-specific cellular genes that play an important role in JEV entry into two highly JEV-susceptible human non-neuronal and neuronal cells. Candidate host factors will be functionally characterized to assess their potential role in promoting JEV entry, using a variety of innovative molecular biology, cell biology, and biochemical techniques. The outcomes of this research will (i) provide a unique opportunity to gain a complete understanding of how JEV-host cell interactions occur at the level of JEV entry, (ii) shed new light on the cell/tissue tropism and pathogenesis of JEV, and possibly other closely related encephalitic flaviviruses, and (iii) provide new targets for the development of novel antiviral interventions capable of inhibiting the early steps of JEV infection.